This study assesses and compares the influence of silver nanoparticles (AgNPs) and silver nitrate (AgNO3) on the fatty acid composition, pigments, and growth indices of Chlorella vulgaris. Toxicity testing was carried at the estimated and/or above predicted environmental concentrations of AgNPs and AgNO3. AgNO3 treatments impaired the population growth of C. vulgaris about 2–183 times more than the respective AgNPs ones. The pigments displayed a concentration-dependent decrease in response to both forms of silver; however, AgNO3 displayed higher severity to the pigments than AgNPs. In exposure to 10 μg L−1 AgNO3, the contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid, respectively, demonstrated a reduction of about 5, 3, 4, and 4 times when compared with the same respective concentration of AgNPs. Total amounts of saturated (∑SFA), monounsaturated (∑MUFA), and polyunsaturated (∑PUFA) fatty acids as well as the ratio of unsaturated to saturated ones (Unsat./Sat.) displayed somewhat similar-concentration responses. ∑SFA exhibited a hormesis response, and ∑MUFA, ∑PUFA, and Unsat./Sat. did a decreasing trend with increasing concentration of AgNPs and AgNO3. Myristoleic acid, nervonic acid, and eicosadienoic acid revealed the highest sensitivity. Pearson analysis illustrated the highest correlation among myristoleic acid, eicosenoic acid, and nervonic acid as well as among palmitic acid, stearic acid, palmitoleic acid, and oleic acid. Taken together, AgNPs and the released ions could disrupt physiological health state of microalgae through perturbation in the fatty acid composition (especially MUFAs and PUFAs) and other macromolecules. These types of bioperturbations could change the good health state of aquatic ecosystems.