Obstructive nephropathy (ON) secondary to the congenital hydronephrosis (HN) is one of the most common causes of chronic kidney disease in children. Neither currently used imaging techniques nor conventional laboratory parameters are sufficient to assess the onset and outcome of this condition; hence, there is a need to prove the usefulness of newly discovered biomarkers of kidney injury in this respect. The purpose of the study was to assess the urinary excretion of alpha-GST, pi-GST, NGAL, and KIM-1 and the serum level of NGAL in children with congenital unilateral hydronephrosis secondary to ureteropelvic junction obstruction. The results were evaluated in relation to severity of HN, the presence of ON, relative function of an obstructed kidney, and the presence of proteinuria. The study comprised 45 children with HN of different grades and 21 healthy controls. Urinary and serum concentrations of biomarkers were measured using specific ELISA kits. Urinary biomarker excretions were expressed as a biomarker/creatinine (Cr) ratio. Patients with the highest grades of HN showed significantly increased values of all measured biomarkers, whereas those with the lowest grades of HN displayed only significant elevation of urinary alpha-GST and the serum NGAL. Urinary NGAL positively correlated with percentage loss of relative function of an obstructed kidney in renal scintigraphy. In patients with proteinuria, significantly higher urinary alpha-GST excretion was revealed as compared to those without this symptom. The ROC curve analysis showed the best diagnostic profile for urinary alpha-GST/Cr and NGAL/Cr ratios in the detection of ON. In conclusion, the results of the study showed that urinary alpha-GST and NGAL are promising biomarkers of ON. Ambiguous results of the remaining biomarkers, i.e., urinary pi-GST and KIM-1, and serum NGAL level may be related to a relatively small study group. Their utility in an early diagnosis of ON should be reevaluated.