This clinical study was conducted to quantify endotoxins and cultivable bacteria in teeth with pulp necrosis and apical periodontitis before and after chemomechanical preparation with 2.5% sodium hypochlorite (NaOCl) and to investigate the possible correlation of endotoxin and cultivable bacteria with the presence of clinical symptomatology. Twenty-four root canals were selected. Samples were collected before (s1) and after chemomechanical preparation (s2). Culture techniques were used to determine the colony-forming unit. A limulus amebocyte lysate (LAL) assay was used to quantify endotoxins (lipopolysaccharide, LPS). LPS and bacteria were detected in 100% of the initial samples (s1), with a median concentration of 139 endotoxin units/mL and 2.64 × 105 colony-forming units/mL, respectively. Higher levels of LPS were found in teeth with clinical symptomatology (p < .05). At s2, mean endotoxin reduction of 59.99% and mean bacterial load reduction of 99.78% were found. Our findings indicated that chemomechanical preparation with 2.5% NaOCl was moderately effective against bacteria but less effective against endotoxins in root canal infection. Furthermore, a statistically significant association was found between higher levels and clinical symptomatology.