Programmed cell death ligand 1 (PD-L1) seems to rely on close relations between neoplastic and immune cells in the tumor microenvironment. Tumor to stroma ratio (TSR) has been associated with prognosis in different malignancies. The aims of this exploratory investigation were to analyze for the first time the: (i) association between TSR, PD-L1 expression and other clinical−pathological features in laryngeal squamous cell carcinoma (LSCC) biopsies and paired surgical specimens; (ii) prognostic and predictive role of TSR and PD-L1. TSR, PD-L1 expression (in terms of combined positive score CPS), and other clinical−pathological features were analyzed in biopsies and surgical specimens of 43 consecutive LSCC cases. A CPS < 1 evaluated on surgical specimens was associated with a low TSR (stroma rich) on both biopsies and surgical specimens (p = 0.0143 and p = 0.0063). Low TSR showed a significant negative prognostic value when evaluated on both biopsies and surgical specimens (HR = 8.808, p = 0.0003 and HR = 11.207, p = 0.0002). CPS ≥ 1 appeared to be a favorable prognostic factor (HR = 0.100, p = 0.0265). The association between bioptic and surgical specimen TSR and PD-L1 expression should be further investigated for a potential impact on targeted treatments, also with regard to immunotherapeutic protocols.