Recommendation of the use of a specific antibiotic for the treatment of bacterial diseases mostly depends on the result of antimicrobial susceptibility testing. Standardization is important to achieve a successful treatment. For example, factors such as incubation period, pH of the medium, concentration of specific antibiotic in the disc and …, may affect the ultimate result. In order to examine the effect of temperature and light over a period of about four years on the antimicrobial activity of different antibiotic discs (chloramphenicol, colistin, enrofloxacin, erythromycin, farazolidone, oxytetracycline, flumequine, lincospectin, tiamulin and tylosin), standard discs were kept under various environmental conditions (temperatures of -12oC, +6oC, +26oC and +37oC, light and dark). At certain time intervals, the antimicrobial activities of discs were estimated by measuring the inhibition zone of Bacillus subtilis growth cultured in Mueller Hinton agar. Comparison of the average zones of inhibition showed a decrease in antibiotic activity of discs with time. However, the storage conditions were less effective in this respect although several antibiotic discs were less affected in cold than warm conditions.