This archive contains the complete hardware, firmware, and software resources required to reproduce and operate the Motilitometer platform. The 3D_files directory includes all mechanical design files for both vertical and horizontal configurations of the system. It provides STL files for 3D printing (camera housing, light guide, sample holder, LED driver enclosure, etc.), as well as full CAD assemblies in STEP and Fusion 360 (F3D) formats. The Motilitometer_software directory contains the full Python-based acquisition and analysis pipeline. It includes modules for dense optical flow, single-cell tracking, video recording, webcam control, light synchronization, and automated sequence processing. A command-line interface (motilitometer.py), documentation (README.md), and a demonstration dataset (demo_vertical.mp4) are provided to facilitate reproducibility and testing. The Motilitometer_light_driver directory provides the complete firmware for the ESP8266-based LED driver, including source files for PWM control, MQTT communication, web interface management (ESPUI), and callback handling. A README file explains compilation and deployment using the Arduino IDE. Together, these resources enable full replication, modification, and extension of both the mechanical and computational components of the Motilitometer system.