Abstract The effects of factors such as weather conditions, leaf age and irrigation water disinfection on the main bacterial genera (total bacterial, Enterobacteriaceae and Pseudomonas) of baby spinach were studied. Culture-dependent and independent quantification techniques were compared. Cultivation was carried out over two consecutive trials in commercial open field divided in two plots: 1) baby spinach irrigated with untreated surface water and 2) baby spinach irrigated with chlorine dioxide (ClO 2) treated water. In all the cases, higher concentrations of bacteria were detected using molecular quantification in comparison with culture dependent techniques. Based on the obtained results, wind speed, solar radiation and relative humidity seem to have an impact on the levels of total bacterial, Enterobacteriaceae and Pseudomonas during cultivation of baby spinach. However, further studies would be needed to confirm this tendency. Water disinfection treatments (ClO 2), when applied to irrigation water, impacted differently the bacterial genera evaluated in the present study. Thus, although no significant effects were observed in total bacterial enumerations of baby spinach irrigated with ClO 2 treated water; significant reductions were detected in Enterobacteriaceae (19%) and Pseudomonas spp. (14%) levels. These results were also confirmed using specific culture-dependent methods. On the other hand, leaf age did not influence the levels of the main bacterial genera of baby spinach. Considering that, a large proportion of foodborne and pathogenic bacteria associated to fresh produce belong to the Enterobacteriaceae family and Pseudomonas genera, reductions in these bacterial groups could be beneficial. However, these groups are very diverse, making difficult to link the measurement of Enterobacteriaceae and Pseudomonas levels with the presence/abundance of potential pathogenic and spoilage microorganisms. Highlights • Culture-dependent and culture-independent methods were compared in baby leaves. • Culture-independent methods gave the highest concentrations. • Leaf age did not affect the major epiphytic bacterial genera. • Water disinfection treatments reduced the Enterobacteriaceae and Pseudomonas spp levels. • Solar radiation and relative humidity showed positive correlation with Pseudomonas spp levels. [ABSTRACT FROM AUTHOR]