In Chiang Mai, Thailand, evidence of a high prevalence of bacterial mastitis with antimicrobial resistance has been observed over the past decade. The development of applicable alternative medicine options is needed to address this resistance situation. The crude extraction of natural resources, such as Japanese pumpkin, should be studied for bacterial activity so as to raise the economic value of each substance. The objective of this study was to explore the antimicrobial activity of Japanese pumpkin by-products against bacteria that has caused dairy mastitis to circulate in the Chiang Mai area. Crude polysaccharides and carotenoids were extracted from Japanese pumpkins. Subsequently, 55 isolates of 10 bacterial species were collected from mastitis milk samples obtained from dairy farms in Chiang Mai during August to October, 2018 were screened for antimicrobial activity using the disc diffusion method. Nine bacterial species displaying inhibition zones were then explored to determine the minimum inhibitory concentrations (MICs). Consequently, 300 mg/ml of crude polysaccharide extracts were not found to be able to inhibit most bacteria. However, the MICs of crude carotenoid extracts ranged from 75 to 300 mg/ml. In summary, by-products of the pumpkin extraction displayed effective antimicrobial activity. Thus, these by-products offer an alternative in preventing the bacteria causing mastitis. This indicates the possibility of developing a prototype of teat dip for the prevention of outbreaks of bacterial infections in the future among dairy cattle. Additionally, this approach could increase the potential value of a large amount of waste produced during the course of industrial processing.