In soft electronics, anisotropic conductive adhesive films (ACFs) are the trending interconnecting approach due to their substantial softness and superior bondability to flexible substrates. However, low bonding pressure (≤1 MPa) and fine-pitch interconnections of ACFs become challenging while being extended in advanced device developments such as wafer-level packaging and three-dimensional multi-layer integrated circuit board assembly. To overcome these difficulties, we studied two types of ACFs with distinct conductive filler sizes (ACF-1: ~20 μm and ACF-2: ~5 μm). We demonstrated a low-pressure thermo-compression bonding technique and investigated the size effect of conductive particles on ACF’s mechanical properties in a customized testing device, which consists of flexible printing circuits and Flex on Flex assemblies. A consistency of low interconnection resistance (<1 Ω) after mechanical stress (cycling bending test up to 600 cycles) verifies the assembly’s outstanding electrical reliability and mechanical stability and thus validates the great effectiveness of the ACF bonding technique. Additionally, in numerical studies using the finite element method, we developed a generic model to disclose the size effect of Au/Ni-coated polymer fillers in ACF on device reliability under mechanical stress. For the first time, we confirmed that ACFs with smaller filler particles are more prone to coating fracture, leading to deteriorated electrical interconnections, and are more likely to peel off from substrate electrode pads resulting in electrical faults. This study provides guides for ACF design and manufacturing and would facilitate the advancement of soft wearable electronic devices.