This study is to investigate the feasibility of our recently developed nonlinear fiberscope for label-free in situ breast tumor detection and lymph node status assessment based on second harmonic generation (SHG) imaging of fibrillar collagen matrix with histological details. The long-term goal is to improve the current biopsy-based cancer paradigm with reduced sampling errors. In this pilot study we undertook retrospective SHG imaging study of ex vivo invasive ductal carcinoma human biopsy tissue samples, and carried out quantitative image analysis to search for collagen structural signatures that are associated with the malignance of breast cancer. SHG fiberscopy image-based quantitative assessment of collagen fiber morphology reveals that: 1) cancerous tissues contain generally less extracellular collagen fibers compared with tumor-adjacent normal tissues, and 2) collagen fibers in lymph node positive biopsies are more aligned than lymph node negative counterparts. The results demonstrate the promising potential of our SHG fiberscope for in situ breast tumor detection and lymph node involvement assessment and for offering real-time guidance during ongoing tissue biopsy.