Antimicrobial resistance (AMR) adversely impacts the prevention and treatment of a wide range of infections and is considered as a serious threat to global public health. Occupational-related AMR is a neglected area of research.To assess exposure to penicillin dust, penicillin active materials, and to report the frequency of penicillin resistance among pharmaceutical workers in Tehran, Iran.A quasi-experimental study was conducted among workers on a penicillin production line in a pharmaceutical company (n = 60) and workers in a food producing company (n = 60). Data were collected via survey, air sampling, and throat swab.The mean overall concentrations of penicillin dust and penicillin active material were 6.6 and 4.3 mg/m3, respectively, in the pharmaceutical industry. Streptococcus pneumoniae (S. pneumoniae) was detected in 45% (27) individuals in the exposed group, 92.6% of which showed penicillin resistance. Resistance was significantly higher among workers in penicillin production line (p = 0.014).High level of AMR among workers in penicillin production line is a health risk for the workers as well as society as a whole through the spread of drug resistant micro-organisms.