Urinary tract infections (UTI) are the most encountered community or hospital-acquired infections (HAI) caused by bacterial pathogens, and about 150 million people are typically affected each year [1, 2]. The microbiological diagnosis of UTI is established by the presence of more than 100,000 colony-forming units (CFUs) per ml of urine accompanied by clinical symptoms of cystitis, pyelonephritis, and asymptomatic bacteria as per the Infectious Diseases Society of America (IDSA) guidelines [3]. UTIs are not restricted to a specific age group. However, factors like age, gender, sexual activity, and the presence of diabetes mellitus (DM) influence the occurrence of UTI [4]. UTI, specifically upper UTI, is common among DM patients because it changes the normal host system and triggers the development of UTI [5]. About 50 to 60% of females are at high risk to have a symptomatic infection in their lifetime, while UTIs occur in the male population in 15 to 20% among whom one-third will have a symptomatic infection requiring antibiotic treatment [6]. Additionally, females are at higher risk of developing UTI than males because of the normal flora of the vagina, its proximity to the anus, sexual intercourse, pregnancy, obstructing lesions, and oestrogen deficiency [7]. Nevertheless, the prevalence of UTI in children is lower than in adult patients and the low rate of increase over the period was reported after 20 years of study (study period 2000–2020) by Boon and colleagues [8]. Bacteria are major causative agents of UTI, especially gastrointestinal bacteria that infect by contaminating the area surrounding the rectum and scattering to the bladder [9]. Among them, Escherichia coli is the most common causative agent, responsible for about 75–85% of infections among Enterobacteriaceae. Other bacteria causing UTI include Pseudomonas species, Enterococcus species, Proteus species, Klebsiella species, Corynebacterium, Neisseria, Coagulase Staphylococcus species, Non-Coagulase Staphylococcus and Streptococcus species. Their geographical distribution varies from one region to another [10]. UTI severity is subject to the virulence, pathogenicity of the bacteria, and susceptibility of the host [11]. So far studies indicate single bacterial species is a cause of UTI rather than more species [12]. Recently, UTIs are difficult to treat due to causative agents showing withstanding effects against antibiotics and limited choice of drugs [13]. The antimicrobial resistance (AMR) developed in uropathogenic bacteria is due to indiscriminate use of antibiotics, poor diagnosis, lack of research, and society’s social-economic status, leading to increased morbidity [14]. In addition, uropathogenic bacteria are the reservoirs of the virulence genes such as immune suppressors (shiA, sisA, sisB, PAI), adhesins (fimH, fimP, traT, kpsMTII, pap), siderophores (iucD, iutA, aer, chuA), and toxins (hlyA, sat, cnf-1); [15] and AMR genes such as Extended-spectrum β-lactamases (ESBLs) encoding blaCTX−M, Klebsiella pneumoniae carbapenemase encoding blaKPC, Metallo-β-lactamases (MBL) encoding blaNDM [16, 17]. The presence of such AMR genes in uropathogens leads to multi-drug resistant bacteria (MDR), which poses a challenge to healthcare settings [18, 19]. MDR uropathogens were reported in the Democratic Republic of Congo (DRC), and it is an alarming condition for the healthcare system of DRC [20]. It is a concern to get in-depth information about the susceptibility of uropathogens and dissemination AMR of the same to fight against the MDR in DRC. However, so far limited data is available on the aetiology of UTI and antimicrobial susceptibility patterns of uropathogenic bacteria in DRC. The successful treatment and prevention of further complications of UTI are achieved by proper empirical treatment [21]. The World Health Organization (WHO) identified surveillance as one of the five strategic priorities of the global and national AMR action plans. When the WHO launched the Global Antimicrobial Resistance Surveillance System (GLASS, https://www.who.int/glass) in 2015, most countries did not have good quality AMR data. To measure the regional AMR burden and generate quality data, WHO supported countries to establish and enhance national AMR surveillance [22]. Therefore, knowledge of the current antibiotic susceptibility patterns of the major bacterial pathogens responsible for UTIs is important to permit the optimal choice for empiric treatment. This study aims to provide the most up-to-date data on the aetiology, and evaluation of AMR patterns of major pathogens responsible for UTI in Butembo, DRC.