pyfastx is a Python C extension library and command-line tool for efficient parsing and random access of FASTA and FASTQ files. It builds SQLite3-based indexes to enable fast sequence retrieval without excessive memory usage, supporting both plain and gzipped files. The core functionality is implemented in C using kseq.h for parsing and zran.c for gzip indexing, with a Python wrapper providing high-level classes (Fasta, Fastq, Sequence, Read) and key collections for filtering and sorting. The included CLI tool offers subcommands for indexing, statistics, splitting, format conversion, subsequence extraction, and sampling. The project includes comprehensive benchmarks comparing against tools like pysam, pyfaidx, and biopython, and is cited in peer-reviewed literature.