To obtain a reliable description of spider communities, robust sampling protocols are crucial. However, it remains unclear if descriptions of spider communities in tropical habitats require both day and night sampling. Here we tested whether sampling both day and night in high and low vegetation strata would lead to better diversity estimates of spider communities than sampling at only one period of the day. We determined spider taxonomic diversity in a network of 12 plots in French Guiana along a vegetation gradient. We found high alpha diversity of spiders as expected for a tropical area at every site. We showed strong differences in spider alpha and beta diversity between high and low vegetation strata, while they were similar between day and night sampling. Our results suggest that collecting spiders at only one period is sufficient to describe the diversity of spider communities across land use types in the neotropics.