Freshwater macroalgae produces a wide range of bioactive compounds, and interest in utilizing its biomass is growing rapidly. Meanwhile, exploiting renewable sources for biomass collection could lead to more sustainable biotechnological chains. The aim of this study was to investigate Cladophora glomerata biomass from Lithuanian rivers as a potential raw material for biotechnology. For this reason, phenolic compound profiles, antioxidant activity and pigment concentrations were determined in macroalgal biomass samples collected from the following four Lithuanian rivers: Dubysa (B1), Šventoji (B2), Nevėžis (B3) and Jūra (B4). The highest total phenolic compound content was determined in B3 (1.32 mg GAE/g). Three phenolic acids were identified, namely gallic (12.94–35.13 µg/g), p-hydroxybenzoic (23.97–29.05 µg/g) and p-coumaric (1.79–6.46 µg/g). The results indicate significant C. glomerata antioxidant activity; the highest reducing power reached 0.737 AU (B3), the total antioxidant content was 1.47 mg Trolox/g (B3), DPPH and ABTS radical scavenging was 11.09% (B3) and 97.86% (B1) and FRAP activity 20.86 µmol/L (B3). The content of pigments ranged from 0.56-0.74, 0.39–0.57, 0.17–0.23 to 0.11–0.17 mg/g in chlorophyll a, b, carotenoids, and lutein, respectively. To conclude, C. glomerata macroalgal biomass may have the potential to act as a functional raw material, as several groups of bioactive compounds and antioxidant activities were observed.