Bovine mastitis is one of the most important diseases affecting dairy herds, yet information on the distribution of causative pathogens remains limited. This study aimed to describe the prevalence and spatio-temporal patterns of mastitis-causing microorganisms in dairy herds from the southeastern region of Brazil. Milk samples (n = 60,863) submitted to three diagnostic laboratories in Minas Gerais between 2009 and 2021 were analyzed using classical bacteriological methods. Descriptive analyses, statistical tests for association, odds ratios, and time series decomposition were performed, and geographic distribution was assessed at the municipality level. 

Of the processed samples, 71.6% showed microbial growth, predominantly Gram-positive bacteria. The most frequent pathogens were coagulase-negative staphylococci (22.2%), Staphylococcus aureus (19.4%), and Streptococcus agalactiae (11.3%). Coinfections occurred in 10.3% of samples, with the combination of S. aureus and S. agalactiae being the most common. Coagulase-negative staphylococci and Corynebacterium spp. acted as protective factors against multiple pathogens, showing reduced odds of co-occurrence with major pathogens. No significant monthly seasonality was detected. However, when grouped by rainfall pattern, Gram-negative organisms, particularly Escherichia coli (OR = 1.52; 95% CI: 1.32–1.76; p < 0.001) and Klebsiella spp (OR = 1.28; 95% CI: 1.11–1.49; p < 0.001) species, had a higher occurrence during the rainy season, while Streptococcus uberis (OR = 0.91; 95% CI: 0.85–0.98; p < 0.05), Corynebacterium spp (OR = 0.87; 95% CI: 0.82–0.92; p < 0.001), and Prototheca spp (OR = 0.50; 95% CI: 0.36–0.69; p < 0.001)had a lower chance of occurrence. 

The findings reveal high prevalence of contagious pathogens and widespread coinfections, reflecting ongoing challenges in mastitis control in Brazilian dairy herds. The protective effect of minor pathogens and the influence of environmental conditions on Gram-negative infections highlight the multifactorial nature of mastitis epidemiology. These results support the need for improved diagnostic accuracy and targeted control programs adapted to regional conditions.