Tumor spread through airspaces (STAS) is a recently determined pathologic phenomenon of lung cancer with significant prognostic impact. This study aimed to analyze the unexplored correlation between preoperative biopsy procedure and a higher risk of STAS and its impact on STAS-related outcomes in resected stage I non-small cell lung cancer (NSCLC). Does preoperative biopsy procedure affect the risk of STAS and STAS-related outcomes in surgically treated stage I NSCLC? We examined 2,169 patients who underwent surgery for pathologic stage I NSCLC from January 2011 through December 2019 at the Seoul National University Bundang Hospital, a tertiary center in South Korea. Factors including percutaneous needle biopsy (PCNB) and bronchoscopic biopsy were assessed for determining the association between preoperative biopsy procedure and an elevated risk of STAS. In addition, the impact of preoperative biopsy on STAS-related prognosis (recurrence and lung cancer-specific mortality) was evaluated with multivariate Cox regression analyses. STAS findings were positive in 638 of 2,169 patients (29.4%). An insignificant association was found between preoperative biopsy (both PCNB and bronchoscopic biopsy) and STAS. After adjustments for preoperative tumor biopsy, STAS was a significant risk factor for cancer recurrence (hazard ratio [HR], 1.72; 95%CI, 1.20-2.48). Additionally, sublobar resection remained a significant risk factor for recurrence (HR, 3.20; 95%CI, 1.65-6.21) and lung cancer-specific mortality (HR, 12.71; 95%CI, 3.68-43.92) in patients with positive STAS findings. However, this association was insignificant for patients without STAS. Preoperative biopsy was not a significant risk factor for either recurrence and mortality, regardless of STAS positivity. Preoperative biopsy in stage I NSCLC neither was associated with an elevated risk of STAS nor influenced the prognosis related to STAS. Physicians can be less apprehensive about performing preoperative biopsy in relationship to STAS.