This study analyzes the spatial and temporal variability of phytoplankton biomass and taxonomic composition in the surface waters of Todos Santos Bay, a semi-enclosed bay within the upwelling zone of the Baja California peninsula (México). Two methodological approaches (microscopy and chemotaxonomy [CHEMTAX]) were used to describe the variability of some genera and species (observed under the microscope) and the importance of those groups that cannot be observed microscopically. Phytoplankton biomarker pigments were measured with high-resolution liquid chromatography and used as input to run the CHEMTAX software to determine the contribution of the main phytoplankton groups to chlorophyll-a (Chl-a). The microscopic analyses showed that diatoms and dinoflagellates comprised 65 and 33% of the total cells, respectively. Dinoflagellates were most abundant in the inner bay and during cold months; the most frequent and abundant genera were Tripos, Protoperidinium, and Prorocentrum. Among diatoms, the most frequent genera were Cylindrotheca, Guinardia, Navicula, Pseudo-nitzschia, and Chaetoceros. CHEMTAX indicated that, together with diatoms, the flagellates prymnesiophytes and chlorophytes contributed a median of 80% to Chl-a, with chlorophytes as the most important group (29.9%). The high contribution of these flagellates suggests that they are represented by small-sized cells not visible by the light microscopy used. However, CHEMTAX largely underestimated the contribution of dinoflagellates (due to the presence of many mixotrophic species). Hence, our results emphasize the importance of using both techniques for a more detailed evaluation of the taxonomic composition of phytoplankton in this and other coastal regions.