Food handlers' training minimizes foodborne illness. Study was conducted from November 2020 to June 2023 to assess the effectiveness of theoretical and practical trainings on the hygienic meat handling improvement of Haramaya, Awaday, Malk-Rafu and Harar towns' butcheries using Staphylococcus aureus as food safety indicator in Eastern Ethiopia. Two rounds (before- and after- training delivery) swab samples from carcasses, equipment (cutting board, hooks and knife), personnel hands and floor, and water sample were collected from 30 randomly selected butchery shops. A total of 420 samples consisted of 210 before- and 210 after- trainings were collected and examined for S. aureus. Selected S. aureus isolated before the training were tested against eight various antimicrobial agents. Per butchery shop (n=30), demography of one worker per shop was also assessed. Prevalence of S. aureus was 51.4% before- and 11.90% after- the delivery of the training. After the training, significant reduction of S. aureus by 39.53% in overall, by 41.66% in environmental and by 26.70% in meat samples were observed. After the training, S. aureus presence was significantly reduced (P <
0.001) by 6.2 to 10.2 folds at studied towns. Except on meat, personnel hands and floor swabs (P >
0.01), S. aureus were significantly reduced after the training in other sampling locations (P<
0.01). Except for cutting board, odds of S. aureus reductions were by 3.2 to 16.7 folds at all sampling locations. S. aureus was 48.9%, 55.1% and 57.1% before delivery of the training in cattle, goat and sheep meat lines, respectively. However, it becomes 9.5%, 11.5%, 13.3% and 14.3% after the delivery of the training in respective the animals' meat lines. All of the 30 butchery shops were found S. aureus positive for at least one location both before the training (50%) and after the training (46.7%) delivery. Of the 53 tested S. aureus, high susceptibility to gentamicin (100%), chloramphenicol (83.02%), sulfamethoxazole-trimethoprim (69.81%) and erythromycin (60.38%) were observed. Two antimicrobial drugs resistant (67.92%) and multiples of ≥ 3 antimicrobial drugs resistant (30.19%) isolates were also observed. Two isolates showed four antimicrobial drugs classes resistant. In conclusion, this study showed the effectiveness of training in reduction of selected foodborne pathogens. However, significances, sustainability and long-term effects of the training with logistic supply and frequent supervision are still recommended.