Bovine mastitis is characterized by pathological alterations of the mammary tissue and physical and chemical changes in the milk causing large economic losses in the dairy industry. The most common etiological agents include a variety of bacteria that may be either contagious or environmental. Furthermore, antibiotic treatment has lost effectiveness, leading to residues in milk and activation of resistance mechanisms in bacteria. Therefore, improving the knowledge about the variety, prevalence, and antimicrobial susceptibility of mastitis-causing bacteria may assist in the development of new strategies to optimize mastitis control. This study aimed to characterize bacterial species causing mastitis from geographically related dairy plants in southern Chile. To do this, milk samples obtained from milking cows diagnosed with clinical mastitis (n=50) and from apparently healthy animals (n=25) were cultured for microbiological characterization, including species identification and antibiotic susceptibility. Morphological identification and Gram staining showed Gram (+) cocci, Gram (-) bacilli, and Gram (+) bacilli. A large diversity of bacteria was detected in both mastitis and non-mastitis samples. The most frequently isolated species were Corynebacterium spp., Staphylococcus haemoliticus, and Staphylococcus aureus. Besides, Staphylococcus haemoliticus and Escherichia coli showed complete resistance and intermediate antibiotic resistance, respectively. Conclusions: the most prevalent variety of bacterial species associated with mastitis in southern Chile are coagulase-negative staphylococci. Furthermore, sampling the entire herd, provides a broader view of the etiological agents causing bovine mastitis. The results of the present study provide an update regarding etiological agents and sampling management of bovine mastitis in southern Chile.