Ventilator Associated Pneumonia (VAP) is a nosocomial infection contracted by patients on ventilators, where bacteria colonize the upper digestive tract and contaminated secretions are released into the lower airway. This nosocomial infection increases the morbidity and mortality of the patients as well as the cost of treatment. Of late Probiotic formulations are postulated to prevent the colonization of these pathogenic bacteria. In this prospective observational study, we aimed to investigate the effects of probiotics on gut microbiota and their relation to clinical outcomes in mechanically ventilated patients. For this study, 30 patients were recruited (20 probiotic treated and 10 without probiotic treatment) screened from a cohort of 169 patients. Patients in the probiotic group were given a dose of 6 capsules of commercially available probiotic (VSL#3®:112.5 billion CFU/cap) in three divided doses for 10 days. Sampling was carried out after each dose to monitor the temporal change in the gut microbiota composition. To profile the microbiota, we used a 16S rRNA based targeted metagenomic approach and differences among the groups were computed using multivariate statistical analyses. Differences in gut microbial diversity (Bray Curtis & Jaccard distance, p-value < 0.05) among the probiotic treated group and the control group were observed. Furthermore, treatment with probiotics resulted in the enrichment of Lactobacillus and Streptococcus in the gut microbiota of the probiotic-treated groups. Our results demonstrated that probiotics may lead to favourable alteration in gut microbiome characteristics. Future studies should focus on appropriate dosages and frequency of probiotics, which can lead to improved clinical outcomes.