Mycotic mastitis mainly caused by yeasts like Cryptococcus spp. and Candida spp. have been emerging since last decade due to several factors like indiscriminate use of antibiotics, immune suppression, corticosteroid therapy, teat injuries and faulty milking machineries. The fungus, Cryptococcus laurentii is a non-neoformans, encapsulated, basidiomycete, which was earlier considered to be saprophytic and non-pathogenic. Now it is increasingly being reported in humans, especially in the immune compromised patients. In this study, Cryptococcus laurentii was isolated and identified from bovine mastitic milk samples. This probably might be the first report of isolation and identification of Cryptococcus laurentii from mastitic milk sample of buffalo from India to best of our knowledge. On milk culture examination, typical creamy white color colonies were appeared on Sabouraud's Dextrose agar, which on Gram’s staining gave budding yeast cells appearance. India ink staining revealed bright halo of capsules surrounding the yeast cells. All the isolates were positive for urease production and biofilm formation. Further confirmation was done using VITEK 2 compact system (BioMerieux) which was based on their biochemical tests profiles. Molecular confirmation was done by the PCR assay. Isolation and identification of this rare fungus from milk samples in present study raises a potential threat of zoonosis.