Extracellular polymeric substances (EPS) play an important role in wastewater sludge structure and stability, yet measured EPS concentrations depend strongly on the extraction method. This study compared three established protocols (thermal sodium carbonate, thermal sodium chloride, and chemical formamide–NaOH extraction) across activated sludge (AS), aerobic granular sludge (AGS), algal–bacterial granular sludge (ABGS), and an AGS–membrane bioreactor (AGS-MBR). Proteins (PN) and polysaccharides (PS) were quantified in soluble, loosely bound, and tightly bound EPS fractions, with total EPS defined as PN + PS. Sodium carbonate consistently produced the highest apparent PN, PS, and total EPS recoveries across the sludge matrices, while sodium chloride and chemical extraction generally produced lower and comparable recoveries. Because the protocols differed in reagent chemistry, temperature, incubation time, and centrifugation conditions, these differences cannot be attributed to a single extraction parameter. A complementary Escherichia coli experiment showed that adding intact cells to an autoclaved-sludge background altered subsequent polymer recovery during sodium carbonate extraction, suggesting a potential contribution of cellular-associated material; however, PN and PS measurements cannot directly demonstrate cell lysis. Overall, extraction-method selection should consider both recovery performance and potential extraction-induced cellular contributions when interpreting and comparing EPS measurements.