A progressive honey bee population decline has been reported worldwide during the last decades, and it could be attributed to several causes, in particular to the presence of pathogens and parasites that can act individually or in synergy. The health status of nine apiaries located in different areas of the Veneto region (northeast of Italy) was assessed for two consecutive years (2020 and 2021) in spring, during the resumption of honey bee activity, for determining the presence of known (Nosema spp., Varroa mite and viruses) and less known or emerging pathogens (Lotmaria passim and Crithidia mellificae) in honey bees. After honey bees sampling from each of the nine apiaries, Nosema apis, Nosema ceranae, L. passim, C. mellificae, ABPV, CBPV, IAPV, KBV, BQCV, SBV, DWV-A, DWV-B and V. destructor were investigated either by microscopic observation or PCR protocols. The viruses BQCV, SBV, CBPV followed by N. ceranae and L. passim were the most prevalent pathogens, and many of the investigated hives, despite asymptomatic, had different degrees of co-infection. This study aimed to highlight, during the resumption of honey bee activity in spring, the prevalence and spreading in the regional territory of different honey bee pathogens, which could alone or synergistically alter the homeostasis of bees colonies. The information gathered would increase our knowledge about the presence of these microorganisms and parasites in the territory and could contribute to improve beekeepers practice.