On-farm biosecurity is important for preventing the spread of several contagious animal diseases. In this study, biosecurity routines among Swedish farmers with livestock (cattle, pigs, sheep or goats) were examined through questionnaires posted by mail. Moreover, the use of protective clothing among professionals visiting farms, such as animal transporters and veterinarians, were investigated through assessments made by the farmers. Questionnaires were completed, partly or fully, by 518 farmers (overall response rate 34%). Possible associations between biosecurity routines and livestock species, geographic location and herd size were analysed. Large variations in biosecurity routines were found, both within and between groups, and some farms appeared to have a relatively high level of biosecurity. However, <40% of the farmers reported that they provide protective clothing for visitors, and 50% of farmers buying live animals introduced these directly into the herd without prior isolation. In general, a higher level of biosecurity was reported by farmers with herds with only pigs, when compared to farmers with cattle, sheep/goats or mixed species. A higher level of biosecurity was also reported by larger farms compared to hobby farms. Inconsistent biosecurity routines were reported, which was interpreted as a lack of knowledge of how different infections can spread and how this can be prevented. Furthermore, some replies indicated that the farmers perceived the risk of introduction of disease as low. According to the farmers' assessments, the use of protective clothing among professionals visiting farms varied considerably, both among different professions and within the same profession. On average, veterinarians and artificial insemination (AI) technicians got high scores in this assessment, while salesmen, repairmen and animal transporters were reported to seldom use protective clothing. Based on the findings, there is room for improvement of on-farm biosecurity. There is also a need to further investigate the motivators and constraints for altered routines among both Swedish livestock farmers and professionals visiting farms.