Background: Vitamin D plays an important role in maintaining bone health with numerous benefits for extraskeletal health as well. Objectives: We aimed to assess the prevalence of vitamin D deficiency in children (0–18 years old) in a tertiary hospital in Romania between August 2019 and January 2024 and to investigate the role of adequate supplementation in this vulnerable population. Methods: The serum 25(OH)D levels were measured using a chemiluminescence binding assay. Results: A total of 744 participants were included in this study: 396 female (53.23%) and 348 male (46.77%). The serum levels of 25(OH)D ranged between 2.2 and 125.4 ng/mL, with a mean value of 27.4 ng/mL and a median value of 23.5 ng/mL. According to the cutoff values for the definition of vitamin D status (severe deficiency: <10 ng/mL, deficiency: <20 ng/mL, insufficiency: 20–29 ng/mL, and sufficiency: ≥30 ng/mL), the sample consisted of 77 (10.34%) cases of severe deficiency, 221 (29.7%) cases of deficiency, 194 (26.07%) cases of insufficiency, and 245 (32.93%) cases of sufficiency. There were seven cases of hypervitaminosis D with values above 100 ng/mL. The mean values (and corresponding 95% confidence intervals, CIs) were as follows: 27.85 ng/mL [25.95–29.76] in the males, 22.45 ng/mL [25.12–28.82] in the females, 24.82 ng/mL [21.86–27.77] in the spring, 28.62 ng/mL [26.42–30.81] in the summer, 32.30 ng/mL [29.16–35.44] in the autumn, and 24.01 ng/mL [21.57–26.44] in the winter. We observed a notable decline in the serum 25(OH)D levels with age, with 82.08% of the children in the age group above 6 years old having serum 25(OH)D levels below 30 ng/mL. In obese subjects, a higher prevalence of hypovitaminosis D was observed compared to non-obese subjects, with a mean value of 19.54 [17.50–21.57] ng/mL in obese children versus 28.89 [27.39–30.40] ng/mL in normal weight children. Conclusions: In our sample, the mean serum concentration of 25(OH)D was 27.4 ng/mL. Notably, 66.11% of the cases demonstrated varying degrees of hypovitaminosis D, with a significantly higher prevalence of 86.16% observed in the obese group. Healthcare providers should prioritize routine screening for vitamin D levels in pediatric patients with obesity to facilitate timely intervention and personalized supplementation strategies tailored to individual needs