Novel human astroviruses (HAstV) were discovered 10 years ago and have been associated with fatal cases of central nervous system infections. Their role in gastroenteritis is controversial, as they have been identified in symptomatic and asymptomatic subjects. The aim of the study was to investigate novel HAstV in a gastroenteritis case-control study including a pediatric population in Spain over a one-year period. We included stool samples from patients with gastroenteritis and negative results for viruses screened by routine diagnostics, and stool samples of control subjects who sought for a routine medical consultation. All samples were screened by real-time RT-PCR assays for novel HAstV. An additional screening for rotavirus, norovirus GI, GII, sapovirus, classic HAstV and adenovirus was also performed for the control group. Overall, 23/363 stool samples from case patients (6.3%) and 8/199 stool samples from control patients (4%) were positive for ≥1 novel HAstV. MLB1 was predominant (64.5% of positives). Seasonality was observed for the case group (p = 0.015), but not the control group (p = 0.95). No difference was observed in the prevalence of novel HAstV between the case and control groups (OR 1.78, 95% CI 0.68-5.45; p = 0.30). Nevertheless, MLB genome copy numbers/ml of fecal suspension was significantly higher in the control group than in the case group (p = 0.008). In our study, we identified a lack of association between novel HAstV and gastroenteritis in the studied population, which could indicate a potential role of reservoir for children, especially given the higher viral load observed in the asymptomatic group for some of them.