Neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP)-9, and NGAL/MMP-9 complex have been evaluated as diagnostic markers of several cancers, but results for bladder cancer are scanty. We evaluated these proteins in urine and serum of 89 patients with histologically confirmed bladder cancer and 119 cancer-free controls from a case-control study. Urinary concentrations were standardized on creatinine level. The performance of these proteins as cancer biomarkers was evaluated through the receiver operating characteristic (ROC) analysis. Urinary level of NGAL, MMP-9, and NGAL/MMP-9 complex was higher in current smokers, whereas no impact of dietary habits was observed. After adjusting for tobacco smoking, urinary concentration of MMP-9 was independently associated with cancer invasiveness, grading, and histological subtype, with elevated concentrations among T2–T4 and non-papillary bladder cancers. Conversely, NGAL and NGAL/MMP-9 complex were significantly higher in non-papillary than in papillary subtype. The pattern was less clear in serum, but correlation between urinary and serum concentration was poor, especially for Ta/is–T1 tumors. The ROC analysis confirmed that MMP-9 was the best marker (area under the ROC curve (AUC) = 0.68). Performances were much greater for muscle-invasive bladder cancers (AUC = 0.90), with elevated negative predictive values (97 %). The present study suggests that NGAL/MMP-9 pathway is associated with an aggressive phenotype of bladder cancer. The elevated negative predictive value of MMP-9 and NGAL/MMP-9 complex makes them candidate markers of exclusion test for bladder cancer. These proteins may be integrated in the surveillance of bladder cancer, thus diminishing patients’ discomfort and improving compliance.