Acute kidney injury (AKI) following snakebite is common in developing countries and Bothrops genus is the main group of snakes in Latin America. To evaluate the pathogenic mechanisms associated with Bothrops venom nephrotoxicity, we assessed urinary and blood samples of patients after hospital admission resulting from Bothrops snakebite in a prospective cohort study in Northeast Brazil. Urinary and blood samples were evaluated during hospital stay in 63 consenting patients, divided into AKI and No-AKI groups according to the KDIGO criteria. The AKI group showed higher levels of urinary MCP-1 (Urinary monocyte chemotactic protein-1) (median 547.5 vs. 274.1 pg/mgCr; p = 0.02) and urinary NGAL (Neutrophil gelatinase-associated lipocalin) (median 21.28 vs. 12.73 ng/mgCr; p = 0.03). Risk factors for AKI included lower serum sodium and hemoglobin levels, proteinuria and aPTT (Activated Partial Thromboplastin Time) on admission and disclosed lower serum sodium (p = 0.01, OR = 0.73, 95% CI: 0.57–0.94) and aPTT (p = 0.031, OR = 26.27, 95% CI: 1.34–512.11) levels as independent factors associated with AKI. Proteinuria showed a positive correlation with uMCP-1 (r = 0.70, p < 0.0001) and uNGAL (r = 0.47, p = 0.001). FENa (Fractional Excretion of sodium) correlated with uMCP-1 (r = 0.47, P = 0.001) and uNGAL (r = 0.56, p < 0.0001). sCr (serum Creatinine) showed a better performance to predict AKI (AUC = 0.85) in comparison with new biomarkers. FEK showed fair accuracy in predicting AKI (AUC = 0.92). Coagulation abnormality was strongly associated with Bothrops venom-related AKI. Urinary NGAL and MCP-1 were good biomarkers in predicting AKI; however, sCr remained the best biomarker. FEK (Fractional Excretion of potassium) emerged as another diagnostic tool to predict early AKI. Positive correlations between uNGAL and uMCP-1 with proteinuria and FENa may signal glomerular and tubular injury. Defects in urinary concentrations highlighted asymptomatic abnormalities, which deserve further study.