Literature on optimum levels and combinations of bioreactor operating parameters to maximize volatile fatty acids (VFAs) via anaerobic digestion (AD) is still scant and contradictory. This study investigated the interactive impact of digester sludge retention time (SRT), temperature, and pH on optimizing VFAs production and linking reactor performance to acidogenic activities. VFAs production from municipal sludge was maximized in semi-continuous flow acid fermenters (AFs). Batch fermentation tests were utilized for the specific acidogenesis activity assays (SAdA) with a simpler substrate (glucose and acetic acid) and inocula from AFs. Fermenters operated at high pH of 8, low SRT of 2 days, and varying temperatures of 55 and 45 °C had the highest chemical oxygen demand (COD) solubilization and acidification yields. Similar fermenters had the highest VFAs fold increase (1.7–2.2) over the substrate, largest acetic acid contribution to total VFAs (52–58%), and maximum SAdA extent (368–461 mL H2/g VSS inoculum). Maximum hydrogen production rate indirectly represented substrate utilization rate, growth of the acidogens, and hence the specific activities of the acidogenic bacteria in the AFs. These results suggested that batch acidogenic assays could provide quantitative information regarding the presence and activities of cultures in continuously-fed AD utilizing more complex substrates, e.g. municipal sludge. A multiple linear regression model was derived successfully predicting VFAs fold increase of AFs with an R-squared value of 0.9999. The constructed contour plots predicted higher VFAs at relatively high fermenter pH (7.5–8.0), lower SRT (2–2.2 days), and lower temperatures (45–48 °C).