The present study was undertaken to ascertain the quarter-wise comparative prevalence, distribution of etiological agents and drug sensitivity pattern of subclinical mastitis (SCM) in crossbred cows and buffaloes. Milk samples collected from 48 dairy animals (30 Jersey crossbred cows and 18 Graded Murrah buffaloes) were confirmed as subclinical mastitis by using six different indirect screening tests: pH, Electrical Conductivity, Chloride Test, Somatic Cell Count, Modified White Side Test and Modified California Mastitis Test and causative organisms were isolated and subjected to in vitro antibiotic sensitivity test. The overall quarter prevalence of subclinical mastitis in crossbred cows and buffaloes were (49.16%) and (45.83%) respectively. Among quarters, prevalence was higher in right hind quarter, followed by left hind quarter, right fore quarter and left fore quarter in crossbred cows and buffaloes. Among the isolates, Staphylococcus spp. showed the highest (40.68%) frequency, followed by Streptococcus spp. (37.29%), E. coli (13.56%) and Bacillus spp. (8.47%) in crossbred cows. In case of buffaloes Staphylococcus spp., Streptococcus spp., E. coli and Bacillus spp. were isolated from 39.39, 30.31, 18.18 and 12.13% milk samples respectively. Ceftriaxone showed the highest values of sensitivity for subclinical mastitis milk sample cultures of crossbred cows and buffaloes (89.83 and 87.87% respectively). It is concluded that right side quarters were found to be more susceptible for SCM than left side quarters because of farmers hand milking nature in crossbred cows and buffaloes. Staphylococcus spp. was the major pathogen responsible for SCM. Ceftriaxone and enrofloxacin were found to be the most effective drug under in vitro condition against the isolated pathogens in SCM affected crossbred cows and buffaloes.