In recent years, a worldwide decline in the Apis mellifera populations has been detected in many regions, including Spain. This decline is thought to be related to the effects of pathogens or pesticides, although to what extent these factors are implicated is still not clear. In this study, we estimated the prevalence of honey bee colony depopulation symptoms in a random selected sample (n = 61) and we explored the implication of different pathogens, pesticides and the flora visited in the area under study. The prevalence of colony depopulation symptoms in the professional apiaries studied was 67.2% [95% confidence interval (CI) = 54.6–79.8; P < 0.0001]. The most prevalent pathogen found in the worker honey bee samples was Nosema ceranae[65.6%; 95% CI = 52.8–78.3; P < 0.0001], followed by Varroa destructor[32.7%; 95% CI = 20.2–45.4; P < 0.0001] and 97.5% of the colonies infected by N. ceranae were unhealthy (depopulated). Co-infection by V. destructor and N. ceranae was evident in 22.9% (95% CI = 11.6–34.3; P < 0.0001) of the samples and only in unhealthy colonies. Of the 40 pesticides studied, only nine were detected in 49% of the stored pollen samples analysed. Fipronil was detected in only three of 61 stored pollen samples and imidacloprid was not detected in any. Acaricides like fluvalinate, and chlorfenvinphos used to control Varroa mite were the most predominant residues in the stored pollen, probably as a result of their application in homemade formulae. None of the pesticides identified were statistically associated to colony depopulated. This preliminary study of epidemiological factors suggests that N. ceranae is a key factor in the colony losses detected over recent years in Spain. However, more detailed studies that permit subgroup analyses will be necessary to contrast these findings.