Background: Mastitis is a multi-etiological disease. It leads to physical, chemical and bacteriological changes in milk and pathological changes in glandular tissues. In the present study, prevalence of mastitis causing organisms with special reference to Pseudomonas aeruginosa and its antibiotic resistance pattern was studied as Pseudomonas spp. is an important organism in harbouring and transmission of antibiotic resistance in environment.Methods: One hundred and one milk samples from mastitic cattle and buffaloes from farms in and around Ludhiana and mastitis cases presented in veterinary clinical complex, GADVASU, Ludhiana, Punjab, India were collected from August 2018 till April 2019. All the samples were processed for isolation of bacteria using different media viz., BHI, EMB, MLA, Cetrimide Agar, BP Agar, Edward’s medium and Blood Agar. After the isolation, all the organisms were subjected to cultural, morphological, biochemical tests and PCR for confirmation. All these isolates (except Bacillus spp. and Serratia marcescens) were screened for their antimicrobial resistance against 15 commonly used antibiotics viz., chloramphenicol, erythromycin, tetracycline, amoxycillin, co-trimoxazole, ciprofloxacin, gentamicin, cephalexin, ofloxacin, sparfloxacin, gatifloxacin, teicoplanin, azithromycin, vancomycin and doxycycline.Result: Out of a total of 101 samples, 86 samples yielded bacterial growth. Out of these 86 samples, 76 (88.37%) samples had a single bacterial growth whereas rest 10 (11.63%) samples had more than one bacterial growth. It was observed after the organisms were subjected to cultural, morphological, biochemical tests and PCR that, a total of 33 (34.02%) Staphylococcus aureus, 24 (24.74%) Escherichia coli, 16 (16.49%) Klebsiella pneumoniae, 5 (5.15%) Pseudomonas aeruginosa, 2 (2.06%) Streptococcus agalactiae, 12 (12.37%) Bacillus spp. and Serratia marcescens 5 (5.15%) were isolated. It was observed that Pseudomonas aeruginosa showed resistance against most of the antibiotics and was sensitive only for ofloxacin (80%) and gentamicin (40%).