In an attempt to compare the microbial community structure between the sea surface microlayer (SML) and subsurface waters (SSW), we determined the enrichment factors (EF: the ratio of abundance or activity in the SML to abundance or activity in SSW) of 13 biological parameters. Samples were taken at 2 contrasting coastal sites in the Mediterranean Sea, corresponding to a high (Barcelona, Spain) and low (Banyuls-sur-Mer, France) urbanized area. Principal component analysis showed that temporal variability was much higher at Barcelona than at Banyuls, and that the characteristics of the SML and SSW samples were more closely related at Barcelona. At both sites, the SML was weakly enriched in heterotrophic bacteria (on average 1.1-fold), Synechococcus spp. (on average 1.1-fold), and photosynthetic picoeukaryotes (on average 1.5-fold) relative to SSW. In contrast, the SML was considerably enriched in chlorophyll a (chl a) (on average 1.9-fold), phaeophytin a (on average 7.4-fold), autotrophic (on average 6.1-fold) and heterotrophic nanoflagellates (on average 5.1-fold) relative to SSW. Enrichments in bacterial production and culturable bacteria were highly variable. EF were significantly different between the 2 sites only for concentrations of chl a, b, and c and the abundance of autotrophic nanoflagellates, with higher EF at the Barcelona site. Except for autotrophic flagellates, the abundance or activity of the parameters determined in the SML was highly correlated with that determined in SSW, suggesting that enrichment of the SML results mainly from upward transport of microorganisms attached to buoyant particles or bubble scavenging. In contrast, the high contribution of autotrophic nanoflagellates to overall phytoneuston biomass (mean = 26%) is likely due to their rapid colonization of the SML. The high abundances of auto- and heterotrophic nanoflagellates in the SML indicated that these organisms play a key role in the functioning of the microbial food webs at the air–sea interface.