The expression of mastitis and its treatment varies based on type and number of pathogens involved. The studies on therapeutics are thus needed for mixed culture in addition to single bacterial etiology. The current study was aimed to find prevalence and drug susceptibility of single and mixed culture of E. coli, Streptococcus agalactiae, Staphylococcus aureus, and Klebsiella pneumoniae against non-beta lactam antibiotics. Total n=200 milk samples from dairy animals were collected and processed for subclinical mastitis and subsequently put to isolation of selected bacteria. A total of n=6 non-beta lactam antibiotics (clindamycin, erythromycin, levofloxacin, vancomycin, teicoplanin and gentamicin) were tested against E. coli, Streptococcus agalactiae, Staphylococcus aureus, and Klebsiella pneumoniae alone, double, triple and tetra combinations using disc diffusion method. Highest prevalence of single bacterial mastitis was observed in case of E. coli while for mixed infection both E. coli plus S. aureus stood at the top. The mixed culture of S. aureus with others in double combination showed higher zones than that of the single against clindamycin. Similarly, E. coli in double and triple combination showed higher sensitivity responses against levofloxacin. Vancomycin showed least efficacy against most of the double, triple and tetra combinations compared to that of single bacteria. Gentamicin on the other hand remained comparatively effective against single and mixed cultures of bacteria. The study thus concluded highly unpredictable responses of bacteria when tested in combination compared to the one used alone which invites further trials to be conducted on underlying complex mechanisms.