Comparing measurements of the natural sea surface microlayer (SML) and artificial surface films made of Triton-X-100 and oleyl alcohol can provide a fundamental understanding of diffusive gas fluxes across the air–water boundary layers less than 1 mm thick. We investigated the impacts of artificial films on the concentration gradients and diffusion of oxygen (O2) across the SML, the thickness of the diffusive boundary layer (DBL), and the surface tension levels of natural seawater and deionized water. Natural and artificial films led to approximately 78 and 81% reductions in O2 concentration across the surfaces of natural seawater and deionized water, respectively. The thicknesses of the DBL were 500 and 350 µm when natural SML was added on filtered and unfiltered natural seawater, respectively, although the DBL on filtered seawater was unstable, as indicated by decreasing thickness over time. Triton-X-100 and oleyl alcohol at a concentration of 2000 µg L−1 in deionized water persistently increased the DBL thickness values by 30 and 26% over a period of 120 min. At the same concentration, Triton-X-100 and oleyl alcohol decreased the surface tension of deionized water from ~72 mN m−1 to 48 and 38 mN m−1, respectively; 47% recovery was recorded after 30 min with Triton-X-100, although low surface tension persisted for 120 min with oleyl alcohol. The critical micelle concentration values of Triton-X-100 ranged between 400 and 459 µg L−1. We, therefore, suggest that Triton-X-100 resembles natural SML because the reduction and partial recovery of the surface tension of deionized water with the surfactant resembles the behavior observed for natural slicks. Temperature and salinity were observed to linearly decrease the surface tension levels of natural seawater, artificial seawater, and deionized water. Although several factors leading to O2 production and consumption in situ are excluded, experiments carried out under laboratory-controlled conditions are useful for visualizing fine-scale processes of O2 transfer from water bodies through the surface microlayer.