Mexico is one of the world's major poultry producing countries. Two significant challenges currently facing the poultry industry are the responsible and judicious use of antimicrobials, and the potential occurrence of infectious disease outbreaks. For example, repeated outbreaks of highly pathogenic avian influenza virus subtype H7N3 have occurred in poultry since its first detection in Mexico in 2012. Both of these challenges can be addressed through good husbandry practices and the application of on-farm biosecurity measures. The aims of this study were: (i) to assess the biosecurity measures practiced across different types of poultry farms in Mexico, and (ii) to collect information regarding antimicrobial usage. A cross-sectional study was carried out through on-farm interviews on 43 poultry farms. A multiple correspondence analysis was performed to characterize the farms based on their pattern of biosecurity practices and antimicrobial usage. Three clusters of farms were identified using an agglomerative hierarchical cluster analysis. In each cluster, a specific farm type was predominant. The biosecurity measures that significantly differentiated the visited farms, thus allowing their clusterization, were: the use of personal protective equipment (e.g. face masks, hair caps, and eye protection), the requirement for a hygiene protocol before and after entering the farm, the use of exclusive working clothes by staff and visitors, footbath presence at the barn entrance, and the mortality disposal strategy. The more stringent the biosecurity measures on farms within a cluster, the fewer the farms that used antimicrobials. Farms with more biosecurity breaches used antimicrobials considered critically important for public health. These findings could be helpful to understand how to guide strategies to reinforce compliance with biosecurity practices identified as critical according to the farm type. We conclude by providing certain recommendations to improve on-farm biosecurity measures.