Abstract: To understand the toxic effects of environmental concentrations of trimethoprim (TMP) on marine microalgae, the effects of TMP on the growth and bioactive components of Skeletonema costatum and Phaeodactylum tricornutum were explored. The findings revealed that TMP had a stimulating effect on the growth of P. tricornutum, among which the 150 ng/L TMP treatment group had the most significant promotion effect, resulting in a maximum 65% increase in cell density. When TMP concentration was below, it promoted the growth of S. costatum to a certain extent; however, as concentration increased, an inhibitory effect was observed (i.e., hormesis). TMP could significantly promoted the synthesis of photosynthetic pigments in P. tricornutum during the early exposure stages, but as exposure time extended, it significantly inhibited pigment synthesis. Furthermore, TMP partially stimulated the synthesis of total protein in P. tricornutum and promoted total lipid synthesis in P. tricornutum at the early stage of exposure. The total carbohydrate and total lipid levels had a similar trend over time, initially being inhibited and subsequently enhanced. Overall, S. costatum displayed greater sensitivity to TMP compared to P. tricornutum.