The aim of this study was to compare the chemical composition of lesser duckweed (Lemna minor L.) obtained from the natural environment and cultured under laboratory conditions. In the fi rst variant, lesser duckweed was collected from a natural pond (LDN). Subsequently, the plants were cleaned of other aquatic flora and fauna, dried, and ground. In the second variant, lesser duckweed was cultured in an aquarium (LDC) illuminated from 04:00 to 24:00. The water temperature was 25 °C and the pH was maintained in the range of 6.5–7.3. The content of crude protein, fat, and fibre was similar in LDN and LDC, but the proportion of crude ash was higher in LDC. The content of total amino acids was 95.70 and 68.71 g/100 g crude protein in LDC and LDN, respectively. The concentrations of essential amino acids and nonessential amino acids were 43.75 and 51.96 g/100 g crude protein in LDC, respectively, and 32.10 and 36.61 g/100 g crude protein in LDN, respectively. Palmitic and stearic acids were found in higher quantities in LDN than LDC, whereas the oleic acid content was three-fold higher in LDN compared to LDC. Moreover, linoleic and α-linolenic acid concentrations were higher in LDN than LDC. Mineral analysis revealed elevated levels of Ca, Na, and Zn, while P and Mg levels were lower in LDC. Additionally, the levels of δ-, γ-tocopherols and α-tocotrienol were found to be higher in LDC, while δ- and γ-tocotrienols in LDC were below detectable limits. So, the significant influence of growth conditions on the nutrient composition of L. minor was shown. Optimizing growth conditions is pivotal for enhancing lesser duckweed production. It seems that L. minor can be a valuable source of essential nutrients and could serve as a supplementary food source for both domestic animals and humans. Nonetheless, anti-nutritional components, including toxic metals, should be monitored.